Enzymes are used within a wide range of applications in industry, research, and medicine. Through the use of enzymes, industrial processes can be carried out at reduced temperatures and pressures and with less dependence on the use of corrosive or toxic substances. The use of enzymes can thus reduce production costs, energy consumption, and pollution as compared to non-enzymatic products and processes.
An important group of enzymes is the proteases, which cleave proteins. Industrial applications of proteases include food processing, brewing, and alcohol production. Proteases are important components of laundry detergents and other products. Within biological research, proteases are used in purification processes to degrade unwanted proteins. It is often desirable to employ proteases of low specificity or mixtures of more specific proteases to obtain the necessary degree of degradation.
Proteases are also key components of a broad range of biological pathways, including blood coagulation and digestion. For example, the absence or insufficiency of a protease can result in a pathological condition that can be treated by replacement or augmentation therapy. Such therapies include the treatment of hemophilia with clotting factors VIII, IX, and VIIa. In another application, the proteolytic enzyme tissue plasminogen activator (t-PA) is used to activate the body's clot lysing mechanism, thereby reducing morbitity resulting from myocardial infarction. The protease thrombin is used to initiate the clotting of fibrinogen-based tissue adhesives during surgery. Neutrophils produce several antibacterial serine proteases (Gabay, Ciba Found. Symp. 186:237-247, 1994; Scocchi et al., Eur. J. Biochem. 209:589-595, 1992). Proteases also regulate cellular processes through receptor-mediated pathways by proteolytic activation of the cognate receptor (Vu et al., Cell 64:1057-1068, 1991; Blackhart et al., J. Biol. Chem. 271:16466-16471, 1996).
Overproduction or lack of regulation of proteases can also have pathological consequences. Elastase, released within the lung in response to the presence of foreign particles, can damage lung tissue if its activity is not tightly regulated. Emphysema in smokers is believed to arise from an imbalance between elastase and its inhibitor, alpha-1-antitrypsin. This balance may be restored by administration of exogenous alpha-1-antitrypsin.
One family of proteases of particular interest is the serine proteases, which are characterized by a catalytic triad of serine, histidine, and aspartic acid residues. Serine proteases are used for a variety of industrial purposes. For example, the serine protease subtilisin is used in laundry detergents to aid in the removal of proteinaceous stains (e.g., Crabb, ACS Symposium Series 460:82-94, 1991). In the food processing industry, serine proteases are used to produce protein-rich concentrates from fish and livestock, and in the preparation of dairy products (Kida et al., Journal of Fermentation and Bioengineering 80:478-484, 1995; Haard and Simpson, in Martin, A. M., ed., Fisheries Processing: Biotechnological Applications, Chapman and Hall, London, 1994, 132-154; Bos et al., European Patent Office Publication 494 149 A1).
In general, enzymes, including proteases, are active over a narrow range of environmental conditions (temperature, pH, etc.), and many are highly specific for particular substrates. The narrow range of activity for a given enzyme limits its applicability and creates a need for a selection of enzymes that (a) have similar activities but are active under different conditions or (b) have different substrates. For instance, an enzyme capable of catalyzing a reaction at 50.degree. C. may be so inefficient at 35.degree. C. that its use at the lower temperature will not be feasible. For this reason, laundry detergents generally contain a selection of proteolytic enzymes, allowing the detergent to be used over a broad range of wash temperature and pH.
In view of the specificity of proteolytic enzymes and the growing use of proteases in industry, research, and medicine, there is an ongoing need in the art for new enzymes and new enzyme inhibitors. The present invention addresses these needs and provides other, related advantages.